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Herpes simplex virus-1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV-1, 2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV-1 DNA or VZV DNA was detected in PBMCs of 50 age- and sex-matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV- 1 or VZV may be associated with the development of some cases of Meniere's disease.

We have analyzed mutations in the thymidine kinase (TK) gene of varicella zoster virus (VZV) which showed resistance to 5-iodo-2'-deoxyuridine (IDU) and 5-bromo-2'-deoxyuridine (BrDU). Through sequencing of the TK gene, we found three amino acids were exchanged (41 Asn-->Ser, 266 Cys-->Ile, 288 Ser-->Leu). These mutations were not located at either the nucleoside- or the ATP-binding site. This result suggests that the resistance to IDU and BrDU in this particular strain is due to the change in conformation of TK rather than the replacement of amino acids in the binding sites.

To investigate the mechanism of action of 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BV-araU) on varicella zoster virus (VZV) replication, we examined the metabolism of the drug in VZV-infected cells using 14C-labeled BV-araU. [14C]BV-araU was taken up by the cells infected with thymidine kinase-positive (TK+)VZV, but not so much by TK- VZV-infected or mock infected cells. Most of the radioactivity in TK+ VZV-infected cells that were incubated with [14C]BV-araU was recovered from their acid-soluble fraction, and little from their acid-insoluble fraction. By high performance liquid chromatographic assay of the acid-soluble fraction, it was proved that BV-araU was metabolized to its 5'-monophosphate, diphosphate, and triphosphate only in TK+ VZV-infected cells. The radioactivity was not detected in VZV nucleocapsids or in VZV DNA and cellular DNA isolated from TK+ VZV-infected cells, even if BV-araU was added at a 1000 times higher concentration than the 50% inhibitory dose for VZV replication in vitro. Furthermore, it was enzymatically proved that [14C]BV-araU was selectively and effectively phosphorylated to BV-araU monophosphate by VZV TK and that affinity of BV-araU triphosphate for VZV DNA polymerase was the quite strong. From these results, it can be concluded that marked inhibition of VZV replication by BV-araU is due to selective phosphorylation of BV-araU in the TK+ VZV-infected cells and strong inhibition of VZV DNA synthesis by BV-araU triphosphate, without detectable incorporation into VZV DNA.

Nine anti-herpes compounds, idoxuridine (IDU), bromovinyldeoxyuridine (BVDU), bromovinylarabinofuranosylurasil (BVaraU), carbocyclic bromovinyldeoxyuridine (C-BVDU), chloroethyldeoxyuridine (CEDU), acyclovir (ACV), dihydroxypropoxymethylguanine (DHPG), adenine arabinoside (Ara-A), and phosphonoformate (PFA) were examined for their inhibitory activities against the replication of twenty-six newly isolated clinical strains of varicella-zoster virus (VZV) in human embryonic fibroblast cell cultures. The order of (decreasing) efficacy was: BVaraU greater than BVDU greater than or equal to C-BVDU greater than CEDU greater than or equal to IDU greater than ACV greater than Ara-A greater than DHPG greater than PFA. There was little variation in the susceptibility of the 26 VZV strains to each particular compound, except for one strain which was resistant to IDU.

The inhibitory effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) 5'-triphosphate on varicella zoster virus (VZV) DNA polymerase was studied using the parent strain (TK+-VZV) and the mutant strain (TK--VZV). The mutant strain was deficient in thymidine kinase (TK)-inducing activity and resistant to BVDU. In the absence of BVDU, TK--VZV and TK+-VZV induced an equivalent level of viral DNA polymerase activity in human embryo fibroblasts. In the presence of 5 microM BVDU, TK--VZV still induced viral DNA polymerase activity, whereas TK+-VZV failed to do so. BVDU 5'-triphosphate (BVDUTP) was considerably more inhibitory to the TK+- and TK--VZV DNA polymerases than to the cellular DNA polymerases. There were no significant differences in the affinity for dTTP as substrate and the sensitivity to BVDUTP as inhibitor between the TK+- and TK--VZV DNA polymerases. The Km value for dTTP and the Ki value for BVDUTP of the VZV DNA polymerases were 1.43 microM and 0.55 microM, respectively. The inhibitory effect of BVDUTP to VZV DNA polymerase was competitive with respect to the natural substrate.

The four antiherpes compounds acyclovir, adenine arabinoside, bromovinyldeoxyuridine, and phosphonoformic acid showed an additive to synergistic effect with human leukocyte interferon in inhibiting focus formation by three different strains of varicella-zoster virus in human embryonic fibroblasts.

A varicella-zoster virus (VZV) strain resistant to 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) but sensitive to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVdUrd) was isolated. The 2'-deoxythymidine (dThd) kinase of this mutant (Ito) strain was characterized; it was much less efficient in phosphorylating dThd, 2'-deoxycytidine, and BrdUrd than were the dThd kinases from wild-type (CaQu, Kobayashi) VZV strains. The Ito dThd kinase had a markedly decreased affinity for dThd, 2'-deoxycytidine, and BrdUrd but only a slightly decreased affinity for IVdUrd than had the wild-type VZV dThd kinase. BrdUrd was incorporated to a much lesser extent in VZV (Ito strain)-infected cells than wild-type VZV-infected cells, but IVdUrd was incorporated in Ito VZV-infected cells as efficiently as in wild-type VZV-infected cells. While resistant to IdUrd and BrdUrd, the Ito strain was susceptible to inhibitors of de novo thymidylate biosynthesis such as aminopterin.

Twenty antiherpes compounds were compared for their inhibitory activities against five laboratory strains and five clinical isolates of varicella-zoster virus (VZV) in human embryo fibroblast cultures. E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), E-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), and E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) were the most effective. The average values of the 50% inhibitory dose (ID50) for VZV replication were 0.0013, 0.0015, and 0.0024 micrograms/ml, respectively. 1-beta-D-Arabinofuranosyladenine and 9-(2-hydroxyethoxymethyl)guanine were 1.2 and 3.4 times less effective than 5-iodo-2'-deoxyuridine (IDU). The selectivity indexes (ratio of ID50 for host-cell DNA synthesis to ID50 for VZV replication) of BVaraU, IVDU, and BVDU were between 41,000 and 67,000, whereas those of cytosine arabinoside, IDU, and 5-bromo-2'-deoxyuridine were less than 1.1. BVaraU, IVDU, and BVDU were the most potent and the most selective inhibitors of VZV activity. A deoxythymidine kinase deficient mutant of VZV was resistant to most of the compounds tested at concentrations up to 36,000 times greater than the average ID50 for strains that were not deficient.

After infection with varicella-zoster virus, HeLa and human embryo lung cells developed a receptor for the Fc portion of human and rabbit immunoglobulin G. The receptor was detected by both hemadsorption and immunofluorescence, using antibody-coated erythrocytes and heat-aggregated immunoglobulin G. However, sheep erythrocytes sensitized with F(ab')2 of anti-sheep erythrocyte antibody did not adsorb to the receptor. When cell-free varicella-zoster virus was inoculated into the HeLa cell monolayer, the Fc receptor appeared at first 6 h after infection; varicella-zoster virus antigen in the cytoplasm and detectable cytopathic effects appeared later.

The in vitro susceptibility of eight strains of varicella-zoster virus (VZV) to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was examined in human embryonic fibroblasts by the following techniques: inhibition of focus formation by either cell-free VZV (4-day assay) or cell-associated VZV (2-day assay), inhibition of viral antigen formation (2-day assay), and inhibition of viral cytopathogenicity (15-day assay). The 50% inhibitory dose (ID50) of BVDU ranged from 0.001 microgram/ml (2-day assay) to 0.01 microgram/ml (15-day assay). BVDU appeared highly selective in its anti-VZV activity since even at concentrations as high as 100 micrograms/ml, BVDU did not markedly affect the viability of the host cells. The ID50 of BVDU for VZV was comparable to that of IVDU (E-5-(2-iodovinyl)-2'-deoxyuridine). Both drugs inhibited the replication of VZV at a much lower concentration than did other antiviral compounds such as iododeoxyuridine, ethyldeoxyuridine, arabinosylcytosine, arabinosyladenine, phosphonoacetic acid, iododeoxycytidine, and acycloguanosine. BVDU and IVDU were virtually inactive against a thymidine kinase-deficient VZV mutant, suggesting that phosphorylation by the viral enzyme is responsible, at least in part, for the selective anti-VZV activity of the compounds.

The activity and mode of action of the new nucleoside analog (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) against varicella-zoster virus (VZV) were determined. In cell culture, replication of different strains of VZV was inhibited to 50% by 0.4 to 0.7 microM 2HM-HBG, while 685 microM was required to inhibit 50% of the DNA synthesis in uninfected human lung fibroblasts. A thymidine kinase-negative VZV strain was not inhibited by 100 microM 2HM-HBG. Inhibition of VZV replication was not reversible after 7 to 14 days of incubation, depending on the multiplicity of VZV. 2HM-HBG was shown to be selectively phosphorylated by purified VZV thymidine kinase, with an inhibition constant of 32.5 microM. The antiviral activity of 2HM-HBG in cell culture was decreased by the addition of deoxythymidine and deoxycytidine but not by other ribo- or deoxyribonucleosides.

One of the plant extracts, glycyrrhizin (GL) was investigated for its antiviral action on varicella-zoster virus (VZV) in vitro. When human embryonic fibroblast (HEF) cells were treated with GL after inoculation of virus (post-treatment), the average 50%-inhibitory dose (ID50) for five VZV strains was 0.71 mM, and the selectivity index (ratio of ID50 for host-cell DNA synthesis to ID50 for VZV replication) was 30. GL was also effective against VZV replication when HEF cells were treated 24 h before the inoculation (pretreatment). Furthermore, at a concentration of 2.4 mM GL inactivated more than 99% of virus particles within 30 min at 37 degrees C. In combination with other anti-herpes drugs (acyclovir, adenine arabinoside, bromovinyldeoxyuridine, and phosphonoformate) or human native beta-interferon, GL had an additive or slightly synergistic effect on VZV replication. The mechanism of anti-VZV action is still unclear. We postulate that GL inhibits the penetration, uncoating or release of virus particles.

The highly potent and selective anti-DNA virus agent (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] was found to inhibit in vitro the replication of a number of clinical varicella-zoster virus strains within the concentration range of 0.63-5.7 ng/ml. With a mean 50% inhibitory concentration of 1.8 ng/ml and selectivity index of 29000, (S)-HPMPA is one of the most potent and most selective varicella-zoster virus inhibitors discovered to date.

Anticomplement Immunofluorescence was used for antibody titration against varicella-zoster virus (VZV) in 43 patients with peripheral facial palsy. Nine of 31 patients (29%) with Bell's palsy and eight of 12 patients (75%) with Ramsey-Hunt syndrome had anticomplement immunofluorescence antibody titres of greater than or equal to 1/10. On the other hand, none of 14 patients with herpes simplex virus (HSV) infection and 51 healthy adults showed anticomplement immunofluorescence antibody titres of greater than or equal to 1/10. The anticomplement immunofluorescence antibody titre in two patients with Ramsey-Hunt syndrome increased later and decreased sooner than the indirect immunofluorescence antibody titre, becoming undetectable at 66 and 104 days, respectively, after onset of the disease. There was no cross reaction between anti-VZV and anti-HSV antibodies in the patients who showed a positive antibody rise for VZV. As the acute stage of VZV infection is obscure in the patients with peripheral facial palsy without herpes the screening of anticomplement immunofluorescence antibody to VZV at titres greater than or equal to 1/10 may be useful for the diagnosis of VZV infection in patients with peripheral facial palsy.

Inhibition of varicella-zoster virus (VZV) replication by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been examined in vitro under various experimental conditions. The 50% inhibitory dose (ID50) of BVDU for VZV replication in human embryonal fibroblast (HEF) cultures was 0.016 micrograms/ml, if the assay was based on the reduction of visible foci. If the assay was based on the reduction of immunofluorescent foci, the ID50 was 3 times higher. There was no significant difference in ID50, whether the HEF cultures were infected with cell-free or cell-associated VZV. When the HEF cells were infected with VZV at different multiplicities of infection (MOI), the ID50 of BVDU increased in parallel with the increase of MOI. BVDU was normally added immediately after virus infection. However, the addition of BVDU could be delayed until 8 hr after infection without substantial decrease of activity. On the other hand, BVDU did not cause any inhibition of focus formation when it was removed within 8 hr after VZV infection. If removed at 24 or 48 hr after infection, BVDU caused a significant reduction in focus formation. BVDU had no effect on focus formation if added to the HEF cells before virus infection. BVDU was also found to inhibit VZV focus formation in Vero cells, but only at an ID50 that was 5-10 times higher than the ID50 noted in HEF cells.

Infection of human peripheral blood monocytes by varicella-zoster virus (VZV) was investigated. When freshly isolated monocytes of young adult volunteers were infected with cell-free VZV and examined by indirect immunofluorescence, specific antigens appeared at 8 hr and the number of antigen-positive cells reached the maximum between 24 and 48 hr postinfection. The proportion of antigen-positive cells to total cells was similar to that of the permissive control (HeLa cells), while very few infectious centers (IC) of monocytes were formed in comparison with the infected control cells. Monocytes isolated from infants and old persons formed a larger number of IC than those of young adults. Electron microscopic study of VZV-infected monocytes from three young adult volunteers demonstrated that the proportion of VZV particle-positive cells to total cells was similar to that of antigen-positive cells, and most of the particles seen in the nuclei were low in density and lacked a central core. These results suggest that the growth of VZV in human adult monocytes is incomplete. This restriction of VZV growth by monocytes may play an important role in defense against VZV infection.

The incorporation of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) into DNA of varicella-zoster virus (VZV)-infected human embryo fibroblasts was studied, using thymidine kinase-positive (TK+) and thymidine kinase-negative (TK-) VZV strains. [125I]IVDU was taken up by cells infected with TK+ VZV-, but not by TK- VZV- or mock-infected cells. [125I]IVDU was incorporated into both VZV DNA and cellular DNA of TK+ VZV-infected cells. When the cells were exposed to 0.3 microM IVDU, a more marked shift was noted in the buoyant density of viral DNA than of host DNA. In contrast, the DNAs isolated from TK- VZV- or mock-infected cells did not exhibit a detectable incorporation of [125I]IVDU. [125I] IVDU-labeled VZV DNA was purified from the viral nucleocapsids of TK+ VZV-infected cells. Substitution of no more than 0.1-1% of the thymidine residues in the VZV DNA by IVDU seemed to suffice to inhibit the replication of VZV.

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella-zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type-1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone-fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.

A simple and sensitive bioassay method for measuring (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) concentrations in human serum and urine has been established. This method is based on the inhibitory effect of BVDU on varicella-zoster virus (VZV) focus formation in vitro. The minimal concentration of BVDU that could be detected in serum by this method was 0.2 microgram/ml. Following a single oral administration of 250 mg BVDU, serum BVDU concentrations of 1.2-2.2 micrograms/ml were attained 1 hr later; at 5 and 7 hr, serum BVDU levels were below 0.2 microgram/ml. Upon repeated administration of 125 mg BVDU at 8 hr intervals, the serum BVDU concentrations reached 0.7-1.1 microgram/ml at 2 hr after the fourth administration. These concentrations are approximately 300-450-fold higher than the 50% inhibitory dose of BVDU for VZV in vitro. Urinary BVDU concentrations were on average 10 to 20 times higher than the serum BVDU concentrations.